The overall goal of this project is to elucidate the genetic control of the conversion of metanephric mesenchyme into differentiated epithelia of the mammalian kidney. Because WT-1 has been documented to be essential for kidney development, the proposed studies will use the WT1 gene as an entry point to begin identifying the cis-acting regulatory elements and their associated transcription factors that are critical to WT1 expression, and therefore to nephrogenesis. In preliminary studies, the investigator has performed the following: 1) Cloned, restriction mapped, and partially sequenced phage, cosmid, and YAC clones that contain the human and mouse WT1 structural genes (100 kb) as well as substantial amounts (up to 240 and 300 kb) of upstream and downstream sequence; 2) Identified cell lines derived from human embryonic kidney that express WT1 (293, 211.1) as well as non-expressing cell lines that can be used as negative controls; 3) Identified 11 DNase hypersensitive sites at the 5' end of the human gene that may represent binding sites for transfactors. Several of these sites also exhibit tissue-specific hypersensitivity; 4) Identified a region approximately 13 kb upstream of the WT1 structural gene that contained a scaffold attachment region, a DNase HS site, and a region of phylogenetic sequence conservation between human and mouse; 5) Performed reporter gene assays in transfected cells that showed that WT1 was under transcriptional control and identified a novel Sp1 site. However, these studies also showed that regions up to 24 kb upstream of the WT1 transcription start site were insufficient to direct tissue-specific expression; 6) Transfected a YAC containing the mouse WT1 gene into human 293 cells and observed expression of the transfected mouse gene, indicating that the YAC contained elements sufficient for expression; and 7) Generated transgenic mice containing 1.9 kb of upstream sequence linked to lacZ reporter gene. Four transgenic founders were examined, and only ectopic expression was fond. Taken together, these results indicated that WT1 was under transcriptional control and that the regulatory elements that mediated tissue specific expression were contained within the cloned YAC but were not located within the proximal upstream region. The first Specific Aim is to identify cis regulatory elements in the mouse and human WT1 gene that are responsible for WT1 kidney specific regulation. Five complementary approaches will be used including DNase I hypersensitive site mapping, phylogenetic sequence conservation, matrix attachment site assays, transfection of YACs containing the WT1 gene into cultured cells, and transgenic animal studies. The second Aim is to identify, clone, and study the expression of transcription factors that bind to critical WT1 cis regulatory regions. Methods to be used include gel mobility shift assays, DNA footprinting, expression cloning, protein purification, and in situ hybridization.